The present disclosure relates to a method for concentrating sample constituents and for multiplying the nucleic acids present in the sample constituents.
The detection of resistances to antibiotics is gaining increasing importance in infectious disease diagnostics owing to the increasing spread of said resistances, for example MRSA and fluoroquinolone resistance in E. coli and in bacteria which cause tuberculosis, sepsis or pneumonia.
In conventional diagnostics, said resistances are usually established using a culture-based method. However, cell culture-based resistance determination is a time-consuming method, since 2-3 days are required for culture and analysis. By analyzing bacterial DNA, resistances can be determined considerably faster. For this purpose, the DNA of the pathogens must be amplified. Amplifications of this kind can, for example, be achieved by the cooperation of DNA and RNA polymerases (nucleic acid sequence based amplification; NASBA), by polymerization and strand displacement (rolling circle amplification; RCA), or by using ligases (ligase chain reaction; LCR). However, owing to its high specificity and sensitivity, the polymerase chain reaction (PCR) is the most commonly used amplification method. In general, the nucleic acid-containing material is, for this purpose, processed and purified prior to PCR.
The processing protocols for nucleic acids envisage multiple work steps in which cells or viruses are accumulated, for example by centrifugation, and the nucleic acids are isolated by cell lysis and various wash steps.
European patent EP 0389063 B2 describes an established purification method of multiple steps, wherein DNA is bound to a solid silica-containing phase in the presence of chaotropic salts. In said method, the cells are first lysed with a chaotropic buffer. The lysate is contacted with a solid silica-containing phase, to which the DNA binds. Subsequently, the solid phase with the bound DNA is separated from the rest of the lysate and the DNA complex is washed. Lastly, depending on the end application, the DNA is eluted from the solid phase or amplified directly in the bound state.
In contrast, “micro total analysis systems” (μ total analysis system, μ TAS) offer the advantage that individual work steps are combined and automated and, at the same time, reduced to a microscale. The potential of systems of this kind lies primarily in their low power requirements, the fast reaction times and in the reduction of sample and reagent volumes, making possible an analytical laboratory on the scale of a chip (“lab-on-a-chip”).
Such a microfluidic system which allows the isolation and amplification of DNA from aqueous solutions is known from WO200465010. When using said system, the sample is first filtered through a membrane, with the cells remaining on the membrane and, in the next step, being removed with the membrane from the filter chamber. After insertion of the membrane into the actual microfluidic system, the cells are washed, lysed, and subsequently the DNA is amplified, with the membrane being removed from the instrument for the amplification and, afterwards, being reinserted. Finally, the amplified DNA is detected while it flows through a channel.
However, providing a robust μ-TAS for diagnostics is made difficult not only by the many work steps between sample processing and PCR reaction, but also by the fact that it is necessary, for example for the analysis of cell- or virus-containing fluids, to have available for analysis amounts which are substantially larger than only a few drops, since media of this kind often contain only very low cell or virus numbers. For example, in the case of sepsis, only 5 pathogens may be present per ml of blood. Thus, it is necessary in the majority of cases to process relatively large sample volumes (two or more milliliters) so that there is a chance of finding enough cells or viruses in the analyte.
The resulting need to switch from macroscopic sample volumes to the desired microliter amounts in a microfluidic system is a problem.